Genetic Screening of LCA in Belgium: Predominance of CEP290 and Identification of Potential Modifier Alleles in AHI1 of CEP290-related Phenotypes

Clinical evaluation of patients

After identification of the molecular cause, clinical records were revisited, based on a clinical checklist comprising data on visual function, retinal appearance and associated (extra-) ocular features. When possible, ERG, fundus pictures, autofluorescence (AF) images and optical coherence tomography (OCT) were obtained. In case of CEP290-related LCA, neurological (MRI) and nephrological data (kidney ultrasound [US], urinary and blood parameters) were evaluated.

Clinical findings

Extensive ophthalmological data (best corrected visual acuity [BCVA], refraction, ERG, visual fields, color vision testing, fundus aspect both with white light and autofluorescence imaging and the presence of nystagmus, night blindness, photophobia and additional features) as well as associated manifestations in patients with CRB1, RPE65, GUCY2D, AIPL1, CRX, RPGRIP1 and RDH12 mutations are summarized in detail in Supp. Table S3. In addition, Figure 1 depicts several representative fundi from patients with an established molecular diagnosis. An MTS due to midbrain abnormalities with cerebellar vermis aplasia was demonstrated in five patients with JS-LCA/CORS and CEP290 mutations (Figure 2).

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Figure 1

Clinical characteristics of eight LCA patients with an established molecular diagnosis, illustrating characteristic phenotypic features associated with different genotypes. CEP290. A & B: Early and later stage phenotype in right eye (RE) in LCA-3 at age 3 and 18 years respectively; note marbleized aspect of midperiphery at age 3, evolving towards atrophy later; macula stays well-preserved throughout evolution. C & D: Fundus and autofluorescence (AF) image of RE of LCA-25 at age eight years; note concentric hyperautofluorescent ring around macula suggesting a watershed zone between better and more affected retina with probably central area the better; midperipheral retina shows diffuse mottled hyperautofluorescence suggesting widespread outer retinal disease. E & F: Fundus image of RE and infrared image of left eye (LE) of LCA-7 at age 33 and 49 years respectively; note pigment epithelium alterations in the mid- and far periphery of retina but no intraretinal pigmentation, and with fair preservation of macular area at age 33; at age 49 macula is still fairly well-preserved, but outer retinal atrophy and spicular intraretinal pigmentation is now prominent. CRB1.G: Fundus of RE of LCA-3 9a at age 16, showing typical yellowish discoloration of atrophic macula, surrounded by nummular type of intraretinal pigmentation; mild pseudopapilledema and prepapillary paravascular fibrosis also visible, as is peripheral greyish hue of outer retinal atrophy with fine white flecks and nummular pigmentation. GUCY2D. H & I: Fundus and AF image of RE of LCA-55 at age 9 years; fundus is essentially quite normal with only mild pigment epithelium alterations in the retinal periphery; however, AF image shows hyperautofluorescence in central macular area. RPE65. J: Fundus of LE of LCA-49 at age 10 who subsequently underwent gene therapy with AAV2-hRPE65v2 in RE (Maguire et al. 2009); apart from some discrete pigment epithelium alterations fundus is essentially normal; autofluorescence imaging could not be obtained due to lack of lipofuscin accumulation in retinal pigment epithelium (RPE) typical of this type of LCA. AIPL1. K & L: Fundus and AF image of RE of LCA-61 at age 19 years; central macular atrophy with yellowish hue is surrounded by area of better preserved peripheral macula; outer retinal atrophy with spicular intraretinal pigmentation visible in periphery; AF shows black area of atrophic central macula, but is typically not surrounded by hyperautofluorescent ring. RDH12. M & N: Fundus of EORD-7 at age 5 and 19 years respectively; note mild macular RPE changes which become more prominent with age; mild predominantly spicular intraretinal pigmentation also increases with age; however, preservation of patches of normal peripheral retina are most striking feature; these patches remain over time.

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Figure 2

Magnetic resonance imaging (MRI) showing characteristic molar tooth sign (MTS) in five patients with Joubert syndrome/cerebello-oculo-renal syndrome due to mutations in CEP290 (all are axial sections through midbrain). Images organized from left to right; arrows indicate MTS of midbrain present in all due to midbrain malformation with hypoplastic cerebellar vermis and midline cleft (all images are T1 weighted except for panel D which is T2 weighted). A) CORS-1 at age 5 years; B) LCA-JS-1 at age 7 years; C) LCA-JS-2 II-1 at age 14 years; D) LCA-JS-2 II-2 at age 17 years and E) LCA-JS-3 at age 2 years.

Special attention was paid to the CEP290-related ocular phenotype, since this has been described in only a few LCA studies so far. It appeared that this phenotype displays only limited fundus alterations in the first few years of life. In a small subset of patients, no fundus abnormalities were obvious early on, while in the majority a marbleized fundus and/or salt and pepper aspect was seen during the first decade. This aspect further evolved from young adulthood into progressive outer retinal atrophy in the midperiphery with relative sparing of the central macula. Abnormalities of the central macula were absent in our patient cohort, despite the impression that CEP290-related disease is probably of a cone-rod type (based on ERG findings and the occurrence of photophobia). Of note is the presence of a hyperautofluorescent ring around the central macula on AF imaging, observed in four patients starting from the age of six (LCA-2, LCA-3, LCA-7 and LCA-25). Mild intraretinal spicular pigment migration occurred in three patients at an age between 7 and 33 years old (LCA-JS-1, LCA-6 and LCA-7). This aspect became even more pronounced at the age of 49 in patient LCA-7, where a predominant spicular pigmentation was mixed with less frequent intraretinal pigment migration with a nummular aspect. Visual acuity of this group was mostly limited to light perception. In the few patients with better preserved central vision, basic color vision was present and visual fields varied from severely concentrically constricted (LCA-8) to sparing of the central 30° at an age of 49 (LCA-7) (Supp. Table S3).

Extra-ocular features of CEP290-related LCA and potential modifier alleles in AHI1

Several patients with CEP290-related retinal dystrophy showed additional systemic features. Two patients with isolated LCA had several symptoms suggestive of renal dysfunction (LCA-3 and LCA-23, Supp. Table S3). LCA-3 suffered from growth retardation, polydipsia, enuresis nocturna and diurnal incontinence. Kidney US at the age of seven, however, was normal. In LCA-23, kidney US at the age of three revealed increased echogenicity and kidneys without clear cortico-medullar differentiation. Despite this observation, no clinical nephrological manifestations were present at the age of 17. Since the age of onset of end-stage renal disease caused by CEP290 mutations may exceed the age of 20 (Helou et al., 2007; Tory et al., 2007), a close nephrological follow-up of these patients is required. Interestingly, both of these patients carry the recurrent c.2991+1655A>G mutation, which so far has only been reported in LCA patients without any other associated pathology. Of note, kidney US was available for only a subset of patients, and in general performed very early in life, when developing kidney disease might be difficult to detect.

In addition, four patients suffered from recurrent otitis media (OM) (LCA-5, SLS-1, SLS-3 and CORS-1, Supp. Table S3). Although this is common in childhood, it is worth mentioning that it is also a clinical manifestation often seen in primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder of motile cilia (Leigh et al., 2009). In a few cases, PCD with OM was associated with X-linked RP, caused by mutations in RPGR (Shu et al., 2007). Notably, RPGR is a centrosomal protein that interacts with CEP290 (Chang et al., 2006). Moreover, loss-of-function experiments of CEP290 in zebrafish caused developmental abnormalities of the otic cavity (Sayer et al., 2006).

Strikingly, 33% of patients with CEP290-related isolated LCA presented with mental retardation and/or autism, in contrast to only 8% of patients with mutations in the other genes. Subtle brain abnormalities such as broadened lateral ventricles were seen on MRI in some patients. Of note, brain imaging was not available for a subset of patients. Additional neurological manifestations included movement abnormalities (LCA-21), and dyspraxia and balance/coordination problems (LCA-18), the latter of which was also evident in two patients with syndromic CEP290-relatedLCA (SLS-2 and CORS-1) (Supp. Table S3).

Taken together, these extra-ocular manifestations fit well into the broad clinical spectrum of CEP290 mutations, varying from isolated LCA to the lethal MKS. In addition, the CEP290 allelic spectrum is highly complex. Mutations associated with isolated LCA in this study were previously reported in other ciliopathies (Table 1), albeit always in compound heterozygosity with a different mutation in the distinct phenotypes. Moreover, identical CEP290 genotypes can display interfamilial variable expressivity and intrafamilial variation of the neurological phenotype was observed in several families with CEP290-related pathology (Coppieters et al., 2010). The complexity of CEP290-related disease is further illustrated by two cases from this study.

The first one is SLS-1, homozygous for p.Trp7Cys. Valente and coworkers identified the same mutation in patient with CORS, also of Pakistani origin (COR22, II:1). Despite a similar ocular and renal phenotype, both patients significantly differ in their neurological phenotype. A second and even more pronounced example is the variability in both nephrological and neurological involvement in three unrelated patients with the same p.Lys1575X/p.Arg1465X genotype (SLS-2, SLS-3 and CORS-1). Patients SLS-3 and CORS-1 displayed a similar clinical course of renal disease, with renal failure at the age of 16 and 14 years, respectively. In contrast, renal insufficiency in patient SLS-2 was not substantiated until the age of 30 (Supp. Table S3). Neurological signs of these three patients ranged from a mild mental handicap (SLS-2) over severe autism in combination with moderate mental retardation (SLS-3) to severe mental retardation associated with ataxia and a MTS (CORS-1). An MRI was not available for the other two patients, however.

The AHI1 gene was screened as a candidate modifier gene in these three patients. Strikingly, CORS-1, with the most severe nephrologic and neurologic phenotype, carries a heterozygous novel p.Asn811Lys mutation in AHI1, which was absent in the two other patients. Upon screening of AHI1 in five additional patients with CEP290-related disease and neurological involvement, a novel missense variant, p.His758Pro, was identified in LCA-3. Mutations in AHI1 encoding Jouberin are responsible for JS, with retinal involvement in 75% and renal involvement in less than 10% of all AHI1-associated patients (Kroes et al., 2008). Interestingly, both p.Asn811Lys and p.His758Pro affect conserved residues and are located in the predicted WD40-repeat, a domain conserved across all eukaryotes, mediating functions such as vesicular trafficking (Li and Roberts, 2001). Since AHI1 and CEP290 appear to be in the same pathway through their interaction with rab8a, mutations in one of both genes may modify a phenotype caused by the other (Kim et al., 2008; Tsang et al., 2008; Hsiao et al., 2009). Tory and colleagues already suggested a similar potential epistatic effect of CEP290 and AHI1 mutations on phenotypes related to NPHP1, encoding another interactor of AHI1 (Tory et al., 2007; Eley et al., 2008). Strikingly, one of the AHI1 variants they described as a potential modifier for neurological involvement in patients with NPHP1 mutations, p.Arg830Trp, was recently identified as a modifier allele for retinal degeneration in patients with NPHP, independent of a primary NPHP1 mutation (Louie et al., 2010). Of note, four out of seven patients in the study from Tory and coworkers carrying p.Arg830Trp displayed visual impairment, with one blind individual (Tory et al., 2007). The p.Arg830Trp variant might affect AHI1 complex stability/formation (Louie et al., 2010). The variants identified here, assumed to represent a neurological modifier in patients with LCA, might disrupt interactions with other proteins, thereby influencing AHI1 function in other organ systems.

Overall, a molecular diagnosis of CEP290 mutations might have considerable consequences towards the clinical prognosis of an individual. Given the potential involvement of ciliary modifiers and the presence of cilia throughout the whole body, the development of various additional clinical manifestations should be taken into account. As for LCA, both children and (young) adults should have a long-term close clinical neurological and nephrological follow-up, since some features have a later onset.

In conclusion, molecular testing identified mutations in 69% of our LCA cohort, with a major involvement of CEP290. Detailed phenotyping of all patients with a molecular diagnosis revealed novel insights, mainly into the CEP290-related retinal phenotype, which is well documented for the first time in a larger patient group. The variable age-of-onset of the extra-ocular features emphasizes the importance of long-term clinical follow-up of LCA patients with CEP290 mutations. Moreover, the identification of potential modifiers of CEP290-related disease might contribute to a refined prognosis based on a molecular diagnosis. Finally, our findings fit with previous observations suggesting that the phenotype of ciliopathies is most likely determined by the synergistic effect of all variants occurring in the ciliary proteome.